GSM7838998: Blastocyst-Polar_WT_rep2; Mus musculus; miRNA-Seq
Normal blastocysts at E4.5, in vivo paused blastocysts at EDG7.5, and mTORi-treated blastocysts were used for microdissection. Laser-assisted separation of ICM and mural trophoblast was performed using a Hamilton Thorne XYRCOS laser system with power setting to 100% and a pulse length of 300 µs (see Supplementary Video 1). For dissection, blastocysts were fixed between two holding pipettes. While the holding pipettes were carefully pulled apart, cellular connections were cut by successive laser pulses, allowing the separation of the blastocyst into pieces containing ICM or mural trophectoderm. Immediately after separation the tissue fragments were transferred into 7 µl PBS and frozen down on dry ice. Small RNA libraries were prepared using the “TrueQuant small RNA-Seq” kit from GenXPro (Germany) according to the manual.  The method is based on a single-tube protocol for ligation of UMI-containing adapters to RNA molecules followed by Reverse Transcription and PCR48. No size selection of the smallRNAs was performed. The small-RNA was amplified with 12-20 cycles to reach the required amounts for sequencing. Sequencing was performed on an Illumina NextSeq500 instrument with 1 x75 bps.